Method for producing polysaccharide-producing cells

Abstract

To provide a method for efficiently producing cells with high polysaccharide production.SOLUTION: Provided is a method for producing polysaccharide-producing cells, comprising culturing plant cells that secrete polysaccharides outside the cells on a solid medium, in which a metal salt is added to a basal medium so that the final concentration is higher by 5 to 200 mM, to induce a callus.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing polysaccharide-producing cells. [Background technology]

[0002] Many kinds of plants are known to produce polysaccharides. To obtain polysaccharides from such plants, methods such as extraction from seeds, fruits, flowers, stems, trunks, leaves, roots, tubers, or tuberous roots of natural or cultivated plants or tapping are used. However, such natural or cultivated plants are easily affected by the natural environment and weather, and therefore have the disadvantage that the production volume and price are not stable. For this reason, a method is also used in which the cells or tissues of such plants are cultured in a liquid medium to grow the cells or tissues and simultaneously produce polysaccharides and secrete them into the medium.

[0003] Methods for producing polysaccharides by culturing plant cells are advantageous in that they are not affected by the natural environment or weather, allowing for stable production of polysaccharides. There is a demand for technology that can improve the production yield of polysaccharides in such methods.

[0004] For example, in Patent Documents 1 and 2, when plant cells such as callus induced from tuberose of the genus Polyanthus are cultured in a liquid medium, a certain amount of metal salt is made to exist in the liquid medium to improve the production of polysaccharides. Polysaccharide-producing cells are generally used in a method in which callus is induced from a target plant, the callus is multiplied by subculturing to obtain a large number of cells, the multiplied polysaccharide-producing cells are cultured in a large amount of liquid medium to produce polysaccharides, and the polysaccharides secreted outside the cells are finally separated from the cells to obtain the target polysaccharide. In Patent Document 1, metal salts are made to exist in the polysaccharide-producing liquid medium to suppress an increase in the viscosity of the liquid medium. As a result of suppressing an increase in the viscosity of the liquid medium, the diffusion and mixing of the culture solution is improved, and the productivity of polysaccharides is improved. In addition, it is said that the separation of the callus or cells from the supernatant containing the target substance is also facilitated, thereby improving productivity. Furthermore, Patent Document 2 claims that the stress resistance of polysaccharide-producing cells is improved and polysaccharide productivity is increased by adding a metal salt to the liquid medium during subculture. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 5-207888 [Patent Document 2] Japanese Patent Application Publication No. 8-131159 Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have investigated a conventional method for producing polysaccharide-producing cells, in which plant cells that secrete polysaccharides outside the cells are cultured in a solid medium to induce callus, and then the callus is cultured in a liquid medium, and have found that the polysaccharide-producing ability of the obtained polysaccharide-producing cells varies greatly depending on the callus strain from which they are derived, and that there are cells (strains) with low polysaccharide-producing ability. Since high-polysaccharide-producing cells with high polysaccharide-producing ability are preferably used in polysaccharide production by culturing plant cells, a method for efficiently producing high-polysaccharide-producing cells is desired. [Means for solving the problem]

[0007] The present inventors have discovered that by culturing plant cells that secrete polysaccharides extracellularly in a solid medium containing a basal medium to which a specific concentration of metal salt has been added, and inducing callus, it is possible to selectively remove low polysaccharide-producing cells with low polysaccharide-producing ability; in other words, it is possible to efficiently produce high polysaccharide-producing cells with high polysaccharide-producing ability, and it is also possible to shorten the time required to produce polysaccharide-producing cells.

[0008] Specifically, the present invention provides a method for producing polysaccharide-producing cells, which comprises culturing plant cells that secrete polysaccharides extracellularly in a solid medium to which a metal salt has been added so that the final concentration is 5 to 200 mM higher than that of a basal medium, and inducing a callus. The present invention also provides a method for producing a polysaccharide, which comprises culturing in a liquid medium the polysaccharide-producing cells produced by the method for producing a polysaccharide-producing cell. Effect of the Invention

[0009] According to the present invention, it is possible to selectively remove cells with low polysaccharide production, and to efficiently produce cells with high polysaccharide production in a short period of time. By using the cells with high polysaccharide production, it is possible to increase the amount of polysaccharide produced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0011] In the present invention, the term "callus" refers to a cell mass having regeneration potential that is generated by dedifferentiation of plant cells. The formation of callus from plant cells is called "callus induction".

[0012] The "plant cells" used in the present invention are not particularly limited as long as they are of a type that produces polysaccharides, and for example, cells of various plants such as okra of the Malvaceae family, sesame oil plant of the Malvaceae family, carrot of the Umbelliferae family, and mint of the Lamiaceae family can be used. Among these, cells of plants of the genus Polianthes L., and more preferably cells of tuberose (Polianthes tuberosa L.) of the genus Polianthes can be suitably used in the present invention.

[0013] Calli induced from Polyanthus tuberosus are known to produce tuberose polysaccharide solution (TPS), an acidic heteropolysaccharide composed of glucuronic acid, mannose, arabinose, galactose, and xylose, which is useful as a cosmetic ingredient due to its surface smoothing and keratin protecting effects.

[0014] In one aspect, the present invention provides a method for producing polysaccharide-producing cells, which comprises inducing callus from plant cells that extracellularly secrete polysaccharides in a solid medium containing a basal medium to which a metal salt has been added so that the final concentration is 5 to 200 mM higher.

[0015] The solid medium used for callus induction is a solid medium to which metal salts have been added in addition to the metal salts originally contained in the basal medium, and does not include a solid medium to which no metal salts have been added. The solid medium can be prepared by a conventional method by adding a predetermined amount of metal salts, a gelling agent such as agarose, gellan gum, or agar, and other components used as necessary to the basal medium. Here, the basal medium refers to a medium capable of maintaining and growing plant cells. As the basal medium, a medium normally used for culturing plant cells can be used, and a commercially available product can also be used. Examples of basal media include LS (Linsmaier-Skoog) medium, MS (Murasige-Skoog) medium, Gamborg B5 medium (Sigma, G5768), White medium (Sigma, W0876), Chu (N6) medium (Sigma, C1416), DKW medium (Sigma, D6162), Hoagland medium (Sigma, H2395), McCown medium (Sigma, M6774), SH medium (Sigma, S6765), etc. Among them, LS medium or MS medium is preferred.

[0016] Basal media usually contain a certain amount of metal salts. For example, the potassium salt concentrations in basal media are 20.0 mM in LS medium, 20.0 mM in MS medium, 24.7 mM in Gamborg B5 medium, 1.7 mM in White medium, 30.9 mM in Chu(N6) medium, 19.8 mM in DKW medium, 6.0 mM in Hoagland medium, 12.6 mM in McCown medium, and 24.7 mM in SH medium. The sodium salt concentrations in basal media are 0.2 mM in LS medium, 0.2 mM in MS medium, 1.3 mM in Gamborg B5 medium, 3.0 mM in White medium, 0.2 mM in Chu(N6) medium, 0.3 mM in DKW medium, 0.0 mM in Hoagland medium, 0.2 mM in McCown medium, and 0.1 mM in SH medium. The calcium salt concentrations in the basal medium are 3.0 mM in LS medium, 3.0 mM in MS medium, 1.0 mM in Gamborg B5 medium, 1.2 mM in White medium, 1.1 mM in Chu(N6) medium, 9.3 mM in DKW medium, 4.0 mM in Hoagland medium, 3.0 mM in McCown medium, and 1.4 mM in SH medium. The magnesium salt concentrations in the basal medium are 1.5 mM in LS medium, 1.5 mM in MS medium, 1.0 mM in Gamborg B5 medium, 3.0 mM in White medium, 0.8 mM in Chu(N6) medium, 3.0 mM in DKW medium, 2.0 mM in Hoagland medium, 1.5 mM in McCown medium, and 1.6 mM in SH medium. In addition, the total concentrations of metal salts contained in the basal media are 25.0 mM in LS medium, 25.0 mM in MS medium, 28.2 mM in Gamborg B5 medium, 8.9 mM in White medium, 33.2 mM in Chu(N6) medium, 32.8 mM in DKW medium, 12.0 mM in Hoagland medium, 17.6 mM in McCown medium, and 28.0 mM in SH medium.

[0017] The solid medium used for callus induction contains a metal salt in addition to the metal salt originally contained in the basal medium. The concentration of the metal salt added to the basal medium is preferably 5 mM or more, more preferably 8 mM or more, even more preferably 10 mM or more, and preferably 200 mM or less, more preferably 150 mM or less, and even more preferably 100 mM or less, as a final concentration. That is, the metal salt is added so that the metal salt concentration is preferably 5 mM or more, more preferably 8 mM or more, even more preferably 10 mM or more, and preferably 200 mM or less, more preferably 150 mM or less, and even more preferably 100 mM or less, as a final concentration, higher than the concentration of the metal salt originally contained in the basal medium. The concentration range of the metal salt added to the basal medium is preferably 5 to 200 mM, more preferably 8 to 150 mM, and even more preferably 10 to 100 mM, as a final concentration. The metal salt concentration in the solid medium is the total concentration of the metal salt originally contained in the basal medium and the metal salt added to the basal medium, and is preferably 13 mM or more, more preferably 16 mM or more, even more preferably 18 mM or more, and is preferably 235 mM or less, more preferably 185 mM or less, and even more preferably 135 mM or less, as a final concentration. The metal salt concentration range in the solid medium is preferably 13 to 235 mM, more preferably 16 to 185 mM, and even more preferably 18 to 135 mM, as a final concentration.

[0018] Alternatively, the concentration of the metal salt added to the basal medium may be, as a final concentration, preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and preferably 500% or less, more preferably 450% or less, and even more preferably 400% or less, when the concentration of the metal salt originally contained in the basal medium is taken as 100%. That is, the metal salt is added so that the metal salt concentration is preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and preferably 500% or less, more preferably 450% or less, and even more preferably 400% or less, when the concentration of the metal salt originally contained in the basal medium is taken as 100%. The concentration range of the metal salt added to the basal medium may be preferably 20 to 500%, more preferably 30 to 450%, and even more preferably 40 to 400%, when the concentration of the metal salt originally contained in the basal medium is taken as 100%.

[0019] As the metal salt, salts of alkali metals such as sodium and potassium, alkaline earth metals such as calcium, magnesium, and barium, and monovalent to trivalent metals such as iron, zinc, and aluminum, for example, chlorides, sulfates, carbonates, phosphates, and nitrates are used. Specific examples of metal salts include sodium chloride, potassium chloride, calcium chloride, barium chloride, magnesium chloride, iron chloride, zinc chloride, aluminum chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, barium sulfate, zinc sulfate, ferrous sulfate, ferric sulfate, aluminum sulfate, sodium carbonate, potassium carbonate, calcium carbonate, barium carbonate, magnesium carbonate, sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, barium phosphate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, and barium nitrate. Among these, at least one selected from the group consisting of alkali metal salts and alkaline earth metal salts is preferred, at least one selected from the group consisting of sodium salts, potassium salts, calcium salts, and magnesium salts is more preferred, and at least one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride is even more preferred.

[0020] From the viewpoint of callus induction efficiency, it is preferable that a plant hormone is added to the solid medium. The type and concentration of the plant hormone may be appropriately selected and set depending on the plant cells used. Examples of the plant hormone include auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), indoleacetic acid (IAA), and indolebutyric acid (IBA); and cytokinins such as furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), and dimethylaminopurine (2iP). Among them, at least one selected from the group consisting of auxins, or a combination of at least one selected from the group consisting of auxins and at least one selected from the group consisting of cytokinins is preferable, and 2,4-D or NAA alone, a combination of NAA and BA, a combination of 2,4-D and kinetin, a combination of 2,4-D and BA, or a combination of NAA and kinetin is more preferable. The concentration of the plant hormone in the solid medium is, for example, 5×10 when 2,4-D or NAA is used alone. -4 M to 1×10 -7 M is preferably 1×10 -5 M to 1×10 -6 M is more preferred, and when using a combination of NAA and BA, a combination of 2,4-D and kinetin, a combination of 2,4-D and BA, or a combination of NAA and kinetin, the concentration of 2,4-D or NAA is 1×10 -4 M to 1×10 -7 M is preferably 1×10 -4 M to 5×10 -6 M is more preferred, and the concentration of BA or kinetin is 1×10 -5 M to 1×10 -9 M is preferably 1×10 -6 M to 1×10 -7 M is more preferred.

[0021] The solid medium may further contain various known additives such as a carbon source. Examples of the carbon source include glucose, fructose, mannose, xylose, sucrose, rhamnose, fucose, and starch, and oligosaccharides such as sucrose are usually used as the carbon source. The carbon source is usually used at a concentration of 0.5 to 10% by weight, preferably 1 to 7% by weight, and more preferably 3 to 5% by weight.

[0022] The pH of the solid medium is, for example, preferably 4.5 to 7.0, more preferably 5.5 to 6.5.

[0023] In the method for producing polysaccharide-producing cells of the present invention, plant cells that secrete polysaccharides outside the cells are cultured on the solid medium to induce callus. For example, a part of a plant body as a plant cell is placed on the solid medium and cultured, and callus can be induced from the plant cell. The part of a plant body is not particularly limited, and includes leaves, stems, flowers, buds, pericarp, fruits, woody parts, bark, roots, rhizomes, seeds, parts thereof, and combinations thereof, and may be appropriately selected depending on the type of plant. For example, in the case of a plant of the genus Polianthes (Polianthes L.), it is preferable to use a part of a flower, and more preferably to use a petal. The size of the part of a plant body used for callus induction is not particularly limited as long as it is a size that allows callus induction. It is preferable to sterilize the part of a plant body used for callus induction in advance by a conventional method.

[0024] Culture conditions for callus induction may be any conditions that allow callus induction, and may be appropriately adjusted depending on the type of plant, the part of the plant used, the type of medium, etc. For example, the culture temperature is preferably 15 to 35°C, more preferably 20 to 30°C. The culture period is preferably 10 to 120 days, more preferably 30 to 90 days. In addition, the culture is preferably performed in a dark place. Here, a dark place means a dark place that is usually used in plant cell culture, and does not need to be completely dark.

[0025] The callus thus induced is a polysaccharide-producing cell, and can be further grown by culturing in a liquid medium to obtain a large number of polysaccharide-producing cells. Thus, in one embodiment, the method for producing polysaccharide-producing cells of the present invention may further include culturing the induced callus in a liquid medium. Meanwhile, before culturing in a liquid medium, the induced callus may be subcultured using the above solid medium while maintaining the same state, if necessary. Thus, in another embodiment, the method for producing polysaccharide-producing cells of the present invention may further include subculture of the induced callus in the above solid medium, and culturing the subcultured callus in a liquid medium.

[0026] The subculture in the solid medium can be carried out by culturing several times, preferably at 15 to 35°C, more preferably at 20 to 30°C, for preferably 10 to 120 days, more preferably 30 to 90 days. There is no particular limit to the number of subcultures, but it is usually 10 times or less. The period from the start of callus induction to the start of culture in a liquid medium (the day when culture in the solid medium for callus induction is started is counted as the first day, and the day before the day when culture in a liquid medium of the induced or induced callus that has been subcultured in the solid medium is started as the last day) corresponds to the culture period in the solid medium for callus induction at the shortest, and is preferably within 300 days, more preferably within 200 days, and even more preferably within 100 days.

[0027] As the liquid medium for culturing callus, the same basic medium as the solid medium for callus induction can be used. Among these, LS medium or MS medium is preferred. The liquid medium may further contain various known additives such as plant hormones and carbon sources. The type and concentration of plant hormones are related to the productivity of polysaccharides. As the plant hormone, for example, auxins such as 2,4-D, NAA, IAA, and IBA; cytokinins such as kinetin, BA, and 2iP; and gibberellins such as gibberellin A3 (GA3) can be used. Among them, at least one selected from the group consisting of auxins, or a combination of at least one selected from the group consisting of auxins and at least one selected from the group consisting of cytokinins is preferred, and 2,4-D or NAA alone, a combination of NAA and BA, a combination of 2,4-D and kinetin, a combination of 2,4-D and BA, or a combination of NAA and kinetin is more preferred. When 2,4-D or NAA is used alone, the concentration of the plant hormone in the liquid medium is 5×10 -4 M to 1×10 -7 M is preferably 1×10 -5 M to 1×10 -6 M is more preferred, and when using a combination of NAA and BA, a combination of 2,4-D and kinetin, a combination of 2,4-D and BA, or a combination of NAA and kinetin, the concentration of 2,4-D or NAA is 1×10 -4 M to 1×10 -7 M is preferably 1×10 -4 M to 5×10 -6 M is more preferred, and the concentration of BA or kinetin is 1×10 -5 M to 1×10 -9 M is preferably 1×10 -6 M to 1×10 -7 M is more preferred.

[0028] Examples of carbon sources include glucose, fructose, mannose, xylose, sucrose, rhamnose, fucose, and starch, and oligosaccharides such as sucrose are usually used as the carbon source. The carbon source is usually used at a concentration of 0.5 to 10% by weight, preferably 1 to 7% by weight, and more preferably 3 to 5% by weight.

[0029] From the viewpoint of improving polysaccharide productivity, a metal salt may be added to the liquid medium in addition to the metal salt originally contained in the basal medium. The concentration of the metal salt added to the basal medium is preferably 5 mM or more, more preferably 8 mM or more, even more preferably 10 mM or more, and preferably 200 mM or less, more preferably 150 mM or less, and even more preferably 100 mM or less, as a final concentration. That is, the metal salt is added so that the metal salt concentration is preferably 5 mM or more, more preferably 8 mM or more, even more preferably 10 mM or more, and preferably 200 mM or less, more preferably 150 mM or less, and even more preferably 100 mM or less, as a final concentration, compared to the concentration of the metal salt originally contained in the basal medium. The concentration range of the metal salt added to the basal medium is preferably 5 to 200 mM, more preferably 8 to 150 mM, and even more preferably 10 to 100 mM, as a final concentration. The metal salt concentration in the liquid medium is the total concentration of the metal salt originally contained in the basal medium and the metal salt added to the basal medium, and is preferably 13 mM or more, more preferably 16 mM or more, even more preferably 18 mM or more, and is preferably 235 mM or less, more preferably 185 mM or less, and even more preferably 135 mM or less, as a final concentration. The metal salt concentration range in the liquid medium is preferably 13 to 235 mM, more preferably 16 to 185 mM, and even more preferably 18 to 135 mM.

[0030] Alternatively, the concentration of the metal salt added to the basal medium may be, as a final concentration, preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and preferably 500% or less, more preferably 450% or less, and even more preferably 400% or less, when the concentration of the metal salt originally contained in the basal medium is taken as 100%. That is, the metal salt is added so that the metal salt concentration is preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and preferably 500% or less, more preferably 450% or less, and even more preferably 400% or less, when the concentration of the metal salt originally contained in the basal medium is taken as 100%. The concentration range of the metal salt added to the basal medium may be preferably 20 to 500%, more preferably 30 to 450%, and even more preferably 40 to 400%, when the concentration of the metal salt originally contained in the basal medium is taken as 100%.

[0031] Examples of metal salts include the same as those in the solid medium. Among them, at least one selected from the group consisting of alkali metal salts and alkaline earth metal salts is preferred, at least one selected from the group consisting of sodium salts, potassium salts, calcium salts, and magnesium salts is more preferred, at least one selected from the group consisting of sodium chloride, potassium chloride, potassium chloride, calcium chloride, and magnesium chloride is even more preferred, and potassium chloride is even more preferred.

[0032] The callus is cultured in a liquid medium at preferably 15 to 35°C, more preferably 20 to 30°C, by carrying out the culture several times for about 7 to 30 days. The culture method is not particularly limited, but shaking culture is preferred. The number of times of culture (number of subcultures) is not particularly limited, but it is preferred to carry out the culture three or more times. It is not necessary to add metal salts to the liquid medium for subculture every time during subculture, but it is preferred to have metal salts present in the liquid medium for subculture many times in order to improve polysaccharide productivity. In addition, when the callus is subcultured in a liquid medium with a significantly different composition, browning may occur, and the proliferation and polysaccharide productivity of the browned callus are reduced. Therefore, when changing the concentration of metal salts or carbon source in the liquid medium, it is desirable to gradually change the concentration for each subculture.

[0033] There are no particular limitations on the culture apparatus, culture tank, etc. used for culturing callus in liquid medium. Various types of culture tanks can be used, such as vertical drums, horizontal drums, and rotary drums. However, it is preferable to use a culture tank that can be shaken. Culture apparatuses are usually equipped with an agitator, but as long as the liquid medium is designed to circulate inside the culture tank, the installation and use of an agitator is not necessarily required.

[0034] As shown in the Examples below, according to the method for producing polysaccharide-producing cells of the present invention, the callus (polysaccharide-producing cells) induced in a solid medium containing a specific concentration of metal salt in addition to the basal medium has a reduced variation in polysaccharide production ability among callus strains, compared with the callus (polysaccharide-producing cells) induced in a conventional solid medium containing no additional metal salt in the basal medium, and callus strains with low polysaccharide production ability can be selectively removed. That is, according to the method for producing polysaccharide-producing cells of the present invention, high polysaccharide-producing cells with high polysaccharide production ability can be efficiently produced. Thus, the method for producing polysaccharide-producing cells of the present invention can be rephrased as a method for producing or selecting high polysaccharide-producing cells, or a method for removing low polysaccharide-producing cells. Here, low polysaccharide productivity means that the amount of polysaccharide produced is less than 80% of the average amount of polysaccharide produced in the conventional method (for example, the method of Comparative Example 1 in the Examples below), and high polysaccharide productivity means that the amount of polysaccharide produced is 80% or more of the average amount of polysaccharide produced. The amount of polysaccharide produced can be measured by known means such as high performance liquid chromatography. Callus induction from plant cells under high metal salt content conditions usually results in poor callus growth and cell death, and is considered to be unsuitable as a callus induction system. However, it was completely unexpected that cells with high polysaccharide productivity can be efficiently produced by inducing callus in a solid medium containing a specific concentration of metal salt in addition to a basic medium. This is presumably because, in the conventional method, callus was induced from plant cells with low salt stress tolerance, and the induced callus survived thereafter, resulting in the presence of callus with low polysaccharide productivity, whereas in the method of the present invention, callus was selectively induced from cells with high salt stress tolerance, resulting in callus with high polysaccharide productivity. Furthermore, as shown in the Examples below, in the method for producing polysaccharide-producing cells of the present invention, unlike conventional methods for producing polysaccharide-producing cells in which induced callus is subcultured on solid medium (e.g., Comparative Example 1 in the Examples below), the method for producing polysaccharide-producing cells of the present invention allows the induced callus to be cultured in liquid medium without being subcultured on solid medium. Furthermore, in the method for producing polysaccharide-producing cells of the present invention, the period for culturing induced callus in liquid medium can be shortened compared to conventional methods for producing polysaccharide-producing cells (e.g., Comparative Example 1 in the Examples below). Therefore, the method for producing polysaccharide-producing cells of the present invention makes it possible to reduce the time, labor, resources, etc. involved in the production of polysaccharide-producing cells.

[0035] The polysaccharide-producing cells obtained by the method for producing polysaccharide-producing cells of the present invention can be directly cultured as polysaccharide-producing cells under known culture conditions and used for industrial or laboratory polysaccharide production. Thus, in another aspect, the present invention provides a method for producing polysaccharides. The method comprises culturing the polysaccharide-producing cells obtained by the method for producing polysaccharide-producing cells of the present invention in a liquid medium. As the liquid medium for polysaccharide production, it is preferable to use a liquid medium having a composition similar to that of the above liquid medium or a liquid medium suitable for polysaccharide production, and it is more preferable to use a liquid medium containing a metal salt as described above.

[0036] The polysaccharides can be collected from the culture thus obtained by, for example, removing the cells from the culture by centrifugation or filtration, concentrating the culture solution using a rotary evaporator or the like, adding ethanol to the concentrated solution to cause precipitation, and freeze-drying the precipitate. The polysaccharides can be purified according to a conventional polysaccharide purification method. For example, a highly purified product can be obtained by dissolving the crude polysaccharide in water, centrifuging the solution to completely remove insoluble matter, and then subjecting the solution to dialysis, ion exchange chromatography, or gel filtration chromatography. EXAMPLES

[0037] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0038] Comparative Example 1 (1) Solid culture 1 (induction of callus) Tuberose buds were cut and sterilized in 70% ethanol solution for 1 minute, then sterilized in 11-fold diluted Kitchen Bleach (KAO) for 15 minutes, and then washed with sterile water. Petals were cut from the sterilized buds to an appropriate size and inoculated onto solid medium. Linsmeyer-Skoog medium (LS medium) was used as the base medium for the solid medium, and 0.18% by weight of gellan gum and 10% by weight of auxin were added. -5 M of NAA (α-naphthalene acetic acid), 10 as cytokinin -6 The medium contained BA (6-benzylaminopurine) as the M and 3% by weight of sucrose as the carbon source. The pH of this medium was adjusted to 5.7 with KOH (potassium hydroxide), and then sterilized in an autoclave at 121°C for 15 minutes. The culture was carried out in the dark at 26±1°C. After 30 to 90 days of culture, it was confirmed that callus had been induced from each petal. After 90 days of culture, the wet weight of each of the 15 calli was measured and found to be 0.71 g on average.

[0039] (2) Solid culture 2 (passage of callus) The calli obtained by culturing for 90 days in the above manner were separated from the petals. The calli were then subcultured three times under the same conditions for 90 days on new solid medium.

[0040] (3)Liquid culture 1 The basic medium for liquid medium A was LS medium, and 10 -5 M of NAA (α-naphthalene acetic acid), 10 as cytokinin -6The composition of the medium was 6-benzylaminopurine (BA) with M and 3% by weight of sucrose as a carbon source. After adjusting the pH of this medium to 5.7 with KOH, 200 mL was placed in a 500 mL Erlenmeyer flask and sterilized in an autoclave at 121°C for 15 minutes. Callus obtained by solid culture was inoculated into this medium. After 2-4 weeks of shaking culture at 26°C and 90 rpm in the dark, the callus was separated from the liquid medium and subcultured in a 500 mL Erlenmeyer flask containing 200 mL of new liquid medium A. This process was repeated multiple times. Liquid culture 1 was carried out for a total of 5 months or more.

[0041] (4)Liquid culture 2 Liquid medium B was prepared by changing the sucrose concentration of liquid medium A to 5% by weight. 200 mL of liquid medium B was placed in a 500 mL Erlenmeyer flask and sterilized at 121°C for 15 minutes using an autoclave. Callus that had undergone liquid culture 1 was inoculated into this medium. After 2-4 weeks of shaking culture at 26°C and 90 rpm in the dark, the callus was separated from the liquid medium and subcultured in a 500 mL Erlenmeyer flask containing 200 mL of new liquid medium B. This process was repeated multiple times. Liquid culture 2 was carried out for a total of more than 5 months.

[0042] (5)Liquid culture 3 Liquid medium C was prepared by adding potassium chloride (KCl) to the composition of liquid medium B so that the final concentration of KCl was 25 mM higher. 200 mL of liquid medium C was placed in a 500 mL Erlenmeyer flask and sterilized using an autoclave at 121°C for 15 minutes. This medium was inoculated with callus that had undergone liquid culture 2. After 2-4 weeks of shaking culture at 26°C and 90 rpm in the dark, the callus was separated from the liquid medium and subcultured in a 500 mL Erlenmeyer flask containing 200 mL of new liquid medium C. This process was repeated multiple times. Liquid culture 3 was carried out for a total of more than 5 months.

[0043] (6) TPS production culture The plant hormones (NAA and BA) contained in liquid medium C were added at 10 -5The medium was replaced with 2,4-D (2,4-dichlorophenoxyacetic acid) from M and used as liquid medium D. 200 mL of liquid medium D was placed in a 500 mL Erlenmeyer flask and sterilized at 121°C for 15 minutes using an autoclave. 10 g of callus from liquid culture 3 was inoculated into this medium (wet weight) and cultured at 26°C and 90 rpm for 14 days in the dark. The callus was then separated from the liquid medium and subcultured (wet weight) in a 500 mL Erlenmeyer flask containing 200 mL of new liquid medium D (wet weight) 10 g of callus. After culture at 26°C and 90 rpm for 14 days in the dark, the culture supernatant was collected.

[0044] (7) TPS Productivity Evaluation (i) Preparation of a calibration curve After immersing Softcare TP-S (KAO) in a dialysis membrane in ultrapure water and dialyzing, the Softcare TP-S in the dialysis membrane was collected and freeze-dried (freeze-dried TPS). Freeze-dried TPS was dissolved in 0.5M NaCl (sodium chloride) solution to prepare TPS solutions of various concentrations, which were then subjected to high-performance liquid chromatography. A calibration curve was created from the peak area values ​​derived from the obtained TPS and the concentration of the TPS solution. For high-performance liquid chromatography, a Chromaster (Hitachi High-Tech Science) was used as the device, a TSKgel G6000PWXL (Tosoh) was used as the column, and 0.5M NaCl, 0.5% sodium benzoate solution was used as the mobile phase, and an RI (differential refractive index detector) was used for detection.

[0045] (ii) Measurement The culture supernatant collected from the TPS production culture was centrifuged at 3000 rpm for 5 minutes in a centrifuge (himac CF7D2), and the supernatant was collected and mixed with an equal amount of 0.5 M NaCl solution, and filtered and purified using a cellulose acetate filter with a pore size of 0.8 μm (ADVANTEC, 39122181). The filtered and purified liquid was subjected to high performance liquid chromatography under the same conditions as for the preparation of the calibration curve, and the TPS concentration of the collected culture supernatant was calculated from the obtained peak area value and the calibration curve.

[0046] The results of evaluating a total of 22 strains are shown in Table 1. The average TPS concentration was 1.01 g / L. Furthermore, 8 of the 22 strains (36.4%) had low TPS production, with a TPS concentration of less than 80% of the average (0.8 g / L).

[0047] [Table 1]

[0048] Example 1 (1) Solid culture (induction of callus) Callus was induced under the same conditions as in (1) Solid Culture 1 described in Comparative Example 1, except that the sucrose concentration in the solid medium was 5% by weight and KCl was added so that the final concentration of KCl was 25 mM higher. After 30 or 90 days of culture, it was confirmed that callus had been induced from each petal. After 90 days of culture, the wet weight of each of the five calli was measured, and the average was 0.20 g.

[0049] (2)Liquid culture 200 mL of a medium having the same composition as liquid medium C described in Comparative Example 1 (5) was placed in a 500 mL Erlenmeyer flask and sterilized using an autoclave at 121°C for 15 minutes. This medium was inoculated with the callus obtained by callus induction in Example 1 (1). After shaking culture at 26°C and 90 rpm for 2 to 4 weeks in the dark, the callus was separated from the liquid medium and subcultured in a 500 mL Erlenmeyer flask containing 200 mL of fresh liquid medium C. This process was repeated multiple times. Liquid culture was continued for a total of 4 months or more.

[0050] (3) TPS production culture Except for using the callus obtained by liquid culture in Example 1(2), the culture and recovery of the culture supernatant were carried out under the same conditions as those for the TPS production culture described in Comparative Example 1(6).

[0051] (4) TPS Productivity Evaluation The TPS concentration was calculated in the same manner as in Comparative Example 1(7). The results of evaluating a total of nine strains are shown in Table 2. There were no low TPS-producing strains with a TPS concentration of less than 0.8 g / L.

[0052] [Table 2]

[0053] Example 2 (1) Solid culture (induction of callus) Callus was induced under the same conditions as in (1) Solid Culture 1 described in Comparative Example 1, except that KCl was added to the solid medium so that the final KCl concentration was 25 mM higher. After 90 days of culture, it was confirmed that callus had been induced from each petal. After 90 days of culture, the wet weight of the callus of each of the 10 plants was measured and the average value was 0.40 g.

[0054] (2)Liquid culture 200 mL of a medium having the same composition as liquid medium C described in Comparative Example 1 (5) was placed in a 500 mL Erlenmeyer flask and sterilized using an autoclave at 121°C for 15 minutes. This medium was inoculated with the callus obtained by callus induction in Example 2 (1). After shaking culture at 26°C and 90 rpm for 2 to 4 weeks in the dark, the callus was separated from the liquid medium and subcultured in a 500 mL Erlenmeyer flask containing 200 mL of fresh liquid medium C. This process was repeated multiple times. Liquid culture was continued for a total of 4 months or more.

[0055] (3) TPS production culture Cultivation and recovery of the culture supernatant were carried out under the same conditions as those for the TPS production culture in Comparative Example 1(6), except that the callus obtained by liquid culture in Example 2(2) was used as the callus.

[0056] (4) TPS Productivity Evaluation The TPS concentration was calculated in the same manner as in Comparative Example 1(7). The results of evaluating a total of 10 strains are shown in Table 3. There were no strains with low TPS production, with a TPS concentration of less than 0.8 g / L.

[0057] [Table 3]

[0058] Example 3 (1) Solid culture (induction of callus) Callus was induced under the same conditions as in (1) Solid Culture 1 described in Comparative Example 1, except that KCl, sodium chloride (NaCl), calcium chloride (CaCl2) or magnesium chloride (MgCl2) was further added to the solid medium. After 90 or 110 days of culture, it was confirmed that callus had been induced from each petal. The wet weights of the calli harvested after 90 or 110 days of culture are shown in Table 4. In both cases, the amount of calli was less than that in solid culture 1 of Comparative Example 1.

[0059] [Table 4]

[0060] (2)Liquid culture 200 mL of a medium having the same composition as liquid medium C described in Comparative Example 1 (5) was placed in a 500 mL Erlenmeyer flask and sterilized using an autoclave at 121°C for 15 minutes. This medium was inoculated with the callus obtained by callus induction in Example 3 (1). After shaking culture at 26°C and 90 rpm in the dark for 2 to 4 weeks, the callus was separated from the liquid medium and subcultured in a 500 mL Erlenmeyer flask containing 200 mL of fresh liquid medium C. This process was repeated multiple times. Liquid culture was continued for a total of 4 months or more.

[0061] (3) TPS production culture Except for using the callus obtained by liquid culture in Example 3(2), the culture and recovery of the culture supernatant were carried out under the same conditions as those for the TPS production culture described in Comparative Example 1(6).

[0062] (4) TPS Productivity Evaluation The TPS concentration was calculated in the same manner as in Comparative Example 1(7). The results of the evaluation are shown in Table 5. There were no low TPS-producing strains with a TPS concentration of less than 0.8 g / L.

[0063]

Table 5

Claims

1. A method for producing polysaccharide-producing cells, comprising culturing plant cells that secrete polysaccharides extracellularly in a solid medium to which a metal salt has been added so that the final concentration is 5 to 200 mM higher than that of a basal medium, and inducing callus.

2. The method of claim 1 further comprising culturing the induced callus in a liquid medium.

3. 2. The method of claim 1, wherein the metal salt is a salt of at least one metal selected from the group consisting of potassium, sodium, calcium, and magnesium.

4. The method according to claim 1, wherein the solid medium contains at least one plant hormone selected from the group consisting of auxins, or a combination of at least one plant hormone selected from the group consisting of auxins and at least one plant hormone selected from the group consisting of cytokinins.

5. The method of claim 1 , wherein the solid medium contains a carbon source.

6. The method according to claim 1, wherein the plant is a plant of the genus Polyanthus.

7. 2. The method of claim 1, wherein the plant is tuberose (Polianthes tuberosa L.).

8. 2. The method according to claim 1, wherein the polysaccharide is an acidic heteropolysaccharide composed of glucuronic acid, mannose, arabinose, galactose, and xylose.

9. The method according to any one of claims 2 to 8, wherein the period from the start of callus induction to the start of culture in a liquid medium is within 300 days.

10. A method for producing a polysaccharide, comprising culturing the polysaccharide-producing cells produced by the method according to any one of claims 1 to 8 in a liquid medium.